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A MOLASSES BASED FERMENTATION MEDIUM FOR MARINE YEAST BIOMASS

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International Journal of Research in Marine Sciences

Universal Research Publications. All rights reserved

Original Article
A MOLASSES BASED FERMENTATION MEDIUM FOR MARINE YEAST
BIOMASS PRODUCTION
Pathissery J. Sarlin1,2* and Rosamma Philip 1
1
Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology, Fine Arts
Avenue, Cochin-16, Kerala, India.
2
PG and Research Department of Zoology, Fatima Mata National College, Kollam.
*Corresponding author: Sarlin. P.J.
Department of Marine Biology, Microbiology and Biochemistry
School of Marine Sciences, Cochin University of Science and Technology, Fine Arts Avenue, Kochi – 16, Kerala, India
Phone: + 914842368120, Fax: + 914842381120
E-mail- sarlinpoly@yahoo.com
Received 20 June 2013; accepted 05 July 2013
Abstract
Four marine yeasts were used for the study based on their performance in a feeding experiment in Fenneropenaeus indicus
viz.,1. Debaryomyces hansenii (S8) 2. Debaryomyces hansenii (S100), 3.Candida sake (S165) and 4.Candida tropicalis
(S186). Molasses was the most preferred carbon source by the marine yeasts compared to glucose, sucrose and rice water.
Molasses (amount of total sugars 9mg/ml) supplemented with peptone (0.75%), yeast extract (0.5%) and MgSO4 (0.25%)
was found to be favouring maximum growth of the four yeast strains tested. Two yeast strains (S8 & S186) showed their
maximum growth at 30ppt salinity and the other two (S100 & S165) at 25ppt and 20ppt respectively. pH6 was found to be
most favourable for growth. This study shows that molasses supplemented with peptone and yeast extract could be used as
a good production medium for large scale production of yeast biomass.
© 2013 Universal Research Publications. All rights reserved
Key words: Marine yeast, molasses, fermentation medium, salinity, pH.
1. Introduction respectively.
Complex nutrients are preferred in fermentation media as The marine yeasts displayed maximum optical density in
they often support higher yields; and chemically defined the pH range 3-8 and showed an abrupt decline in growth at
media are rarely used due to economic reasons. Of all the NaCl concentration above 5% level. An incubation
medium components used, carbon and nitrogen sources are temperature of 20-250C is often used for the growth of
of particular importance in the medium, since microbial mesophilic yeasts [2, 3]. [4] studied the influence of pH and
cells are composed largely of these elements. The major temperature on the growth and citric acid production of
carbon and nitrogen source of fermentation media are YarrowialipolyticaH222 with glucose as a substrate and
soybean meal, molasses, corn steep liquor, sulphite waste found that the best fermentation conditions were pH 6 and
liquor, cotton seed meal, yeast extract, peptone etc. temperature 300C. Debaryomyces nepalensis, a halo
Calcium chloride, ammonium phosphate and potassium tolerant food spoiling yeast could grow in complex (YEPD)
phosphate are incorporated for enhanced growth. Microbes medium at pH 3.0 to 11.0 in the absence of salt and pH
grow more vigorously on complex media than in mineral 3.0-9.0 in the presence of different concentrations of NaCl
media, because the former contain biosynthetic precursors and KCl [5] . Debaryomyces hansenii is salt tolerant and it
that can be channeled directly into anabolic pathways has been reported that some strains are able to grow in the
reducing the need to produce them and saving metabolic presence of 20% (W/V) NaCl [6] and Zygosaccharomyces
energy. Pharmamedia, molasses, corn steep liquor, sulphite rouxii is recognized for its tolerance of high concentrations
waste liquor are used as fermentation substrates for of sugars [7]. Debaryomyces hansenii, and its anamorph
microbes. [1] tested the optimal growth condition of two Candida formata, are cryotolerant marine yeasts which can
marine yeast strains D. hansenii (Yeast-14) and C. tolerate salinity levels up to 24%, whereas Saccharomyces
austromarina (Yeast-16) and observed that maximal cerevisiae growth is inhibited when salinity reaches 10%.
growth was in the range of 20 - 300C and 20 - 250C The present study is focused on the selection of a cheap and

39 International Journal of Research in Marine Sciences 2013; 2(2): 39-44

suitable substrate for the production of marine yeast
biomass for application in food and feed industry.
2. Materials and methods
2.1Yeasts used for the study
Four marine yeasts (Fig.1) were used for the study based on
their performance in feeding experiment in
Fenneropenaeus indicus viz.,1. Debaryomyces hansenii
(S8) 2. Debaryomyces hansenii (S100) 3. Candida sake
(S165) and 4. Candida tropicalis (S186). These yeast
strains were originally isolated from the Arabian Sea and
maintained in the Microbiology Laboratory of the
Department of Marine Biology, Microbiology and
Biochemistry, Cochin University of Science and
Technology.
Fig.1. Marine yeasts used for the study (100x).
2.2 Preparation of inoculum
The yeast isolates were streaked onto malt extract agar
(malt extract, 30g; mycological peptone, 5g; Agar-Agar,
20g; sea water, 1Litre; pH, 7.6) slants and after incubation
for 3-4 days at room temperature (2820C), the cells were
harvested using sterile physiological saline (0.9% NaCl in
distilled water). The optical density of the cell suspension
was adjusted to 1O.D (approximately 4X10 8 cells/ml) and
this cell suspension was used as inoculum. 1 ml was
inoculated to 100 ml medium.
2.3. Measurement of growth
The yeast growth was estimated by measuring the optical
density at 540nm in a Hitachi Model 200-20 UV Visible
Spectrophotometer.
2.4 Selection of a suitable substrate as Carbon source
for growth
Four different media (M1, M2, M3 and M4) were prepared
incorporating substrates such as glucose, sucrose, rice
water and molasses as Carbon sources. The basal medium
(KNO3, 2g; Seawater,1000ml, pH-5.5) was supplemented
with Glucose, 20g (M1); Sucrose, 20g (M2); Rice water,
100ml (M3); and Molasses, 50ml (M4). 1ml aliquots of the
inoculum were transferred into 100ml media. Incubated at
room temperature (2820C), for 48 hours and the growth
were measured by recording the optical density at 540 nm
in a Hitachi Model 200-20 UV-Visible spectrophotometer.
Based on the results medium containing molasses (M4) was
selected for further study (Fig. 2).
40 International Journal of Research in Marine Sciences 2013; 2(2): 39-44
Fig.2 Growth of marine yeasts in media with different C
sources ( M1 - Glucose , M2 - Sucrose , M3 – Rice water
and M4 - Molasses).
2.5 Optimization of the concentration of molasses in
medium.
Concentration of molasses in the medium was expressed in
terms of total sugars estimated by Anthrone Method [8].
50g of molasses in 100ml seawater was used as the stock.
Media with different concentrations of molasses (1 to
10mg/ml total sugars) were prepared. After sterilization,
the selected marine yeasts were inoculated and they were
incubated at room temperature (2820C) for 48 hours and
growth was estimated at 540nm.
2.6 Optimum peptone concentration
Molasses media with different peptone concentration (0,
0.25, 0.5, 0.75 and 1%) were prepared. Inoculation was
done and the cultures were incubated at room temperature
(2820C). After 48 hours growth was measured at 540nm.
2.7 Optimum yeast extract concentration
Molasses media (M4) with different yeast extract
concentration (0, 0.25, 0.5, 0.75 and 1%) were prepared.
Inoculation was done and the cultures were incubated at
room temperature (2820C). After 48 hours growth was
measured at 540nm.
2.8 Optimum Magnesium sulphate concentration
Molasses media (M4) supplemented with different
concentrations of magnesium sulphate (0, 0.25, 0.5, 0.75
and 1%) were prepared. Inoculation was done and the
cultures were incubated at room temperature (2820C).
After 48 hours growth was measured at 540nm.
2.9 Optimum Calcium chloride concentration
Molasses media with different calcium chloride
concentrations (0, 0.25, 0.5, 0.75 and 1%) were prepared.
Inoculation was done and the cultures were incubated at
room temperature (2820C). After 48 hours growth was
measured at 540nm.
2.10 Optimum Potassium dihydrogen phosphate
concentration
Molasses media supplemented with different
concentrations of potassium dihydrogen phosphate (0, 0.1,
0.2, 0.3 and 0.4%) were prepared. Inoculation was done
and the cultures were incubated at room temperature
(2820C). After 48 hours growth was measured at 540nm.
2.11 Effect of salinity
Molasses medium (M4) was prepared in seawater of
different strength (0, 10, 15, 20, 25, 30, 35 and 40 ppt).
Inoculation was done and the cultures were incubated at 3.3 Supplementation of Peptone and Yeast extract
room temperature (2820C). After 48 hours growth was Even though a significant increase in growth could not be
measured at 540nm. observed with the supplementation of peptone in the
2.12 Effect of pH medium, maximum growth could be obtained at a
Molasses media (M4) with different pH (4,5,6,7,8,9 and 10) concentration of 0.75% (Fig. 4).Considerable increase in
were prepared using various buffers. (Various buffers used growth could be observed when yeast extract was
were Sodium acetate-acetic acid pH-5.0, Tris-Maleic acid introduced into the medium and the growth was found to be
pH-6.0-7.0, Tris-HCl Buffer pH-8.0, NaHCO3-Na2CO3 pH- maximum at 0.5% (Fig. 5).
9.0). All the four yeast strains were inoculated and
incubation was done at room temperature (2820C) for 48
hours.
3. Results
3.1 Selection of suitable Carbon source for media
preparation
Molasses was found to be the best carbon source
supporting maximum growth followed by rice water and
sucrose. Maximum growth was exhibited by Debaryomyces
hansenii (S100) followed by Candida sake (S165). Growth
in molasses medium was found to be almost double of that
found in the other media.
3.2 Effect of molasses concentration on growth
Growth of marine yeasts was found to be influenced by the
concentration of molasses in the medium. Maximum Fig. 5. Growth of yeast strains at various yeast extract
growth was observed at a concentration of total sugars concentrations in the molasses medium. Data are given as
9mg/ml for all the four strains tested. A gradual increase in mean ± SD, n=3.
growth could be observed with the increase of molasses
concentration. However, the presence of total sugars more
than 9mg/ml was found to have adverse effect and resulted
in lesser growth (Fig.3).

Fig. 6. Growth of yeast strains at various magnesium

sulphate concentrations in the molasses medium . Data are
given as mean± SD, n=3.
Fig. 3. Effect of molasses concentration on the growth
of yeast strains. Data are given as mean ± SD, n=3

Fig. 4. Growth of yeast strains at various peptone Fig. 7. Growth of yeast strains at various calcium chloride
concentrations in the molasses medium. Data are given as concentrations in the molasses medium. Data are given as
mean ± SD, n=3 mean ± SD, n=3.

41 International Journal of Research in Marine Sciences 2013; 2(2): 39-44

3.4 Supplementation of Magnesium, Calcium and
Potassium
Generally growth was found to be maximum at a
concentration of 0.25% MgSO4 in the medium (Fig.6). The
optimal calcium chloride concentration for growth was
found to be 0.15% for all the strains (Fig.7). Generally the
optimum potassium dihydrogen phosphate concentration
was 0.3% (Fig.8).
Fig. 8. Growth of yeast strains at various KH2PO4
concentration in the molasses medium. Data are given as
mean± SD, n=3.
3.5 Effect of salinity and pH on growth
A salinity of 30ppt was preferred by S8 and S186 whereas
S100 showed maximum growth in 25ppt and S165 in 20ppt
(Fig.9). Growth was meager at pH 4 and increased
considerably at pH 5 reaching the maximum at pH 6
(Fig.10).
Fig. 9. Effect of salinity on the growth of selected strains.
Data are given as mean ± SD, n=3
Fig. 10. Effect of pH on the growth of selected yeast
strains. Data are given as mean ± SD, n=3.
42 International Journal of Research in Marine Sciences 2013; 2(2): 39-44
4. Discussion
Molasses was found to be the most preferred substrate by
the marine yeasts for growth and a concentration of 9mg/ml
(total sugars) was found to be optimal for all the four
strains. S. cerevisiae had the highest cell viability and
ethanol production in a molasses medium containing 25%
(w/v) total sugars at 350C [9]. Molasses being a waste from
sugar industry, its utilization would be highly economical
for SCP production. Molasses as a crude C source might
have contributed better growth due to the presence of other
nutrients. The results obtained with rice water was not very
promising displaying the nutritional insufficiency of the
medium. In the present study, besides the carbon sources
supplied, the medium contained only KNO3 (N source) and
the nutrients present in the seawater. [10] has reported that
Cane molasses used as C source contain 60% sucrose in
addition to growth promoting components. Cane molasses
(3.5-17.5% w/v total sugar) and yeast powder (1.5-5% w/v)
were used in the formulation of media for the cultivation of
Aspergillus japonicas FCL-119T and Aspergillus niger
ATCC 20611 [11]. They found that lower sugar
concentration produced lower cellular growth.
Carbon sources in fermentation media can be simple or
complex carbohydrates, sugar alcohols or other alcohols,
organic acids, proteins, peptides, amino acids and even
hydrocarbons. These carbon sources are usually used in a
crude form of sugars which include beet and cane molasses,
corn molasses, whey, sulfite waste liquor, cull fruits,
cannery wastes and so forth. Polysaccharides such as
starches are supplied by corn, wheat, rye, milo, rice,
potatoes, sweet potatoes and other agricultural products.
Cellulosic byproducts also are usable as carbon sources, but
they usually require costly saccharification by a procedure
such as acid hydrolysis. Hydrocarbon substrates are
usually a mixture of various hydrocarbon components and
are relatively inexpensive. The pure hydrocarbon
compounds or hydrocarbon fractions, however, are more
costly. Several of the better crude nutritive sources for
fermentation media are themselves complex mixtures of
nutrients, supplying carbon and nitrogen compounds as
well as microbial growth factors. Specific examples are
molasses, corn steep liquor and sulfite waste liquor. The
overall composition of the various molasses differs
according to the specific geographic areas of production.
Beet and Cane molasses are by-products of the sugar
industry and different names are applied to the molasses
depending on the particular step from which it was
recovered. Of these, blackstrap molasses prepared from
sugar cane normally is the cheapest and the most used
sugar source for industrial fermentations. Beet molasses are
produced by procedures resembling those for sugar cane.
However, beet molasses may be limiting in biotin for yeast
growth so that a small amount of cane blackstrap or other
source of biotin should be added for growth of these
microorganisms. Hydrol is a molasses resulting from the
manufacture of crystalline dextrose from corn starch. It
contains approximately 60% sugar, but it also contains a
relatively high salt concentration that must be considered if
this molasses is to be used as a medium component. Cane
molasses is the final run-off syrup from sugar manufacture.
Total residual sugars in molasses can amount to 50-60%
(w/v), of which 60% is sucrose. In addition to sucrose there
are both growth promoting components [10] and inhibitors,
e.g. Hydroxymethyl furfural [12] .
4.1 Supplementation of Peptone and Yeast extract
The optimum peptone concentration was found to be
0.75%. Generally for bacteria a concentration of 0.5 to 1%
is incorporated in media. In the present study also, the
optimal concentration falls within this range. The optimum
yeast extract concentration for growth was found to be
0.5% for all the four strains. [13] reported that the
maximum production of cephalosporin C by Acremonium
chrysogenum was achieved by employing wheat rava with
1% soluble starch and 1% w/w yeast extract at 30 0C. Yeast
growth medium require more amount of yeast extract in it
than that required in bacteriological media. ZoBell’s agar
contains only 0.1% yeast extract, whereas GPY (Glucose
Peptone Yeast extract) used for yeast cultivation contain
1% yeast extract. The presence of hydrolyzed yeast
components would be definitely supporting good growth.
4.2 Supplementation of Calcium, Magnesium and
phosphate
The optimum magnesium sulphate concentration was found
to be 0.25%. [14] observed that the optimal medium for
pullulan production by Rhodotorula bacarum was 8.0%
(w/v) glucose, 2.0% (w/v) soybean cake hydrolysate, 0.5%
(w/v) MgSO4.7H2O, and 0.06% (NH4)2SO4, pH 7.0.
Calcium chloride can control pH of the fermentation
medium and has its’ own effect on growth. In the present
investigation, presence of 0.15% calcium chloride in the
medium was most favourable for growth.
Phosphate can enhance or suppress the production of
growth at different concentrations. According to [15]
vegetative growth of yeasts increased with the initial
phosphate concentration up to 5mM, a further increase of
phosphate showed no significant effect on cell yield. In the
present study the optimum phosphate concentration was
found to be in the range 0.2 to 0.3%.
4.3 Effect of salinity and pH
Among the four strains used for the study, two strains (S8
and S186) showed 30ppt as their optimal salinity for
growth. Other two strains (S100 and S165) showed
maximum growth at 25ppt and 20ppt respectively. Strains
which showed a preference for 30ppt seawater exhibit their
halophilic nature. D. hansenii is salt tolerant and it has been
reported that some strains are able to grow in the presence
of 20% (w/v) NaCl [6, 16]. Similar observations were
made by [17] that the D. hanenii can grow under low water
activity. The greater tolerance of Debaryomyces hansenii
and Saccharomyces hansenii to NaCl at pH 5.0 – 7.0
compared with other pH values has also been reported by
Hobot and [18] . Debaryomyces hansenii is cryotolerant
marine yeast which can tolerate salinity levels up to 24%.
An optimal pH of 6 was found to be very much favourable
for growth by majority of the strains. Environmental pH is
particularly significant in determining the growth of yeasts
[19, 20]. Most yeasts grow within the range of pH 3.0-7.0
[21, 22, 23]. The effects of temperature, pH and NaCl
concentration on the growth of 13 strains of yeasts
representing five genera: Debaryomyces, Pichia,
43 International Journal of Research in Marine Sciences 2013; 2(2): 39-44
Zygosaccharomyces, Candida and Saccharomyces revealed
that there was a synergistic effect between NaCl and pH at
lower temperatures. The pH of seawater is about 7.8 and
therefore marine yeasts usually prefer a higher pH for
growth compared to those isolated from fruits and
terrestrial environments, which prefer a pH of 4-5.5 for
maximal growth [24]. The optimum process variables that
supported maximum phytase production by a marine yeast
KodamaeaohmeriG3 were statistically determined as
23.0% NaCl and initial pH 6.3 [25].
[5] noticed that the growth of the food spoilage yeast D.
nepalensis in NaCl and KCl was completely inhibited when
temperature was maintained at extreme conditions (-1 and
+1). At extreme pH of 3.6, specific growth was 0.29 and
0.37/h for NaCl and KCl, respectively exhibiting the
yeast’s preference for acidic conditions for growth.
Optimization of the process parameters using RSM clearly
showed the strong interaction effect between the pH,
temperature, and salt concentration on the growth of food
spoilage yeast D. nepalensis.
[26] found that a strain of Candida sp. S27 acts as a good
immunostimulant and antioxidant when applied as a dietary
supplement in Penaeus monodon. [27] observed that
Candida sp. S27 promote growth in Penaeus monodon
while supplied through diet at 5% level.
Molasses being a byproduct from sugar industry is a cheap
source of Carbon for fermentation industry. Marine yeast
prefers molasses for its growth and thereby mass
production of yeast single cell protein can be achieved by
using molasses as the basal medium. Supplementation of
Nitrogen source and minerals enhanced the yeast biomass
production. Molasses supplemented with peptone, yeast
extract and minerals can be used as a good fermentation
medium for large scale production of yeast biomass for
application in food and feed industry.
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